Emerging role of miRNAs in the regulation of ferroptosis

Ferroptosis is a kind of cell death which has distinctive features differentiating it from autophagy, necrosis and apoptosis. This iron-dependent form of cell death is described by an increase in lipid reactive oxygen species, shrinkage of mitochondria and decrease in mitochondrial cristae. Ferroptosis is involved in the initiation and progression of many diseases and is regarded as a hotspot of investigations on treatment of disorders. Recent studies have shown that microRNAs partake in the regulation of ferroptosis. The impact of microRNAs on this process has been verified in different cancers as well as intervertebral disc degeneration, acute myocardial infarction, vascular disease, intracerebral hemorrhage, preeclampsia, hemorrhagic stroke, atrial fibrillation, pulmonary fibrosis and atherosclerosis. miR-675, miR-93, miR-27a, miR-34a and miR-141 have been shown to affect iron metabolism, antioxidant metabolism and lipid metabolism, thus influencing all pivotal mechanisms in the ferroptosis process. In the current review, we summarize the role of microRNAs in ferroptosis and their involvement in the pathetiology of malignant and non-malignant disorders.


Introduction
As a newly recognized kind of cell type, ferroptosis is associated with accumulation of large amounts of iron accumulation and lipid peroxidation during the process of cell death . This concept has been firstly proposed by Dixon et al. as an iron-dependent way of cell death described by an increase in lipid reactive oxygen species (ROS) (Dixon et al., 2012). It has several distinctive features that distinguishes this mode of cell death from autophagy, necrosis and apoptosis (Dixon et al., 2012;Xie et al., 2016). Lack of swelling of the cytoplasm and cell organelles and absence of cell membrane splitting differentiate ferroptosis from necrosis. Moreover, absence of cell shrinkage and chromatin condensation, lack of establishment of apoptotic bodies and absence of cytoskeleton breakdown differentiate ferrptosis from apoptosis. Finally, the typical closed bilayer membrane organizations which are produced during autophagy are never seen in ferroptosis . From a morphological point of view, ferroptosis is characterized by shrinkage of mitochondria and decrease in mitochondrial cristae, features which are not seen in other types of cell death (Yagoda et al., 2007;Yang and Stockwell 2008;Dixon et al., 2012). Ballooning is a specific phenotype acquired by cells during ferroptosis, defined by the establishment of a clear, rounded cell chiefly consisting of empty cytosol (Battaglia et al., 2020). Notably, during the process of ferroptosis the cell membrane is not affected, the size of nucleus is not changed, and chromatin is not condensed. However, the intracellular content of glutathione (GSH) is depleted and activity of glutathione peroxidase 4 (GPX4) is decreased. Thus, the metabolism of lipid peroxides by GPX4 is impaired. Subsequently, oxidation lipids of by Fe 2+ in a Fenton-like mode results in production of massive quantities of ROS, which induces ferroptosis (Yang and Stockwell 2008;Angeli et al., 2014). Ferroptosis is regulated by several genetic factors, most of them being involved in modulation of iron homeostasis and lipid peroxidation .
Ferroptosis happens through two main routes, i.e., the extrinsic and the intrinsic pathways. While the former is called the transporter-dependent pathway, the latter is regulated by enzymes. This process is initiated by a redox imbalance between synthesis of oxidants and antioxidants due to the abnormalities in the expressions and activities of several redox-active enzymes that synthetize or detoxicate free radicals and products of oxidation of lipids. Therefore, ferroptosis is finely regulated at several phases. This ROS-associated kind of cell death is related with two major biochemical features, i.e., iron buildup and lipid peroxidation .
Ferroptosis is involved in the initiation and progression of many diseases and is regarded as a hotspot of investigations on treatment of disorders . Recent studies have shown that microRNAs (miRNAs) partake in the regulation of ferroptosis. The impact of miRNAs on this process has been verified in different cancers as well as non-malignant conditions. In the current review, we summarize the role of miRNAs in ferroptosis and their involvement in the pathetiology of malignant and non-malignant disorders.
miRNAs effect on ferroptosis in cancers miRNAs are a group of small-sized non-coding transcripts that can specifically bind with their target transcripts and induce its degradation or inhibit its translation. Through regulating several biological processes, these transcripts have fundamental roles in the process of development and cellular homeostasis (Liu et al., 2014). They can also affect expression of genes which has role in iron metabolism (Zolea et al., 2017). An experiment in melanoma cell lines has shown the role of miR-9 regulation of ferroptosis through influencing expression of GOT1. This miRNA could suppress expression of GOT1 through binding to 3′-UTR of GOT1 transcript. This binding leads to reduction of erastin-and RSL3-associated ferroptosis. On the other hand, miR-9 silencing could increase the response of neoplastic cells to erastin and RSL3. Moreover, the impact of miR-9 suppression in accumulation of lipid ROS and induction of ferroptosis can be abolished by suppression of glutaminolysis process .
The Kaposi's sarcoma herpes virus (KSHV)-encoded miRNAs have been shown to enhance expression of xCT which encodes a membrane-associated amino acid transporter. This process is mainly accomplished via inhibition of BACH-1, a modulator of transcription which recognizes antioxidant response elements within promoter regions. Enhancement of xCT expression by KSHV miRNAs has an important role in promotion of cell permissiveness for KSHV infection and protection of infected cells from reactive nitrogen species-induced cell death (Qin et al., 2010).
miR-17* is another miRNA that participate in the pathogenesis of cancers through influencing ferroptosis. This miRNA can inhibit activity of a number of enzymes participating in mitochondrial antioxidant pathways, namely manganese superoxide dismutase (MnSOD), GPX2 and thioredoxin reductase-2 (TrxR2). Forced up-regulation of miR-17* in PC-3 cells has decreased expressions of these antioxidant proteins through binding to their 3′-UTR. Cumulatively, miR-17* can inhibit prostate carcinogenesis via suppression of mitochondrial antioxidant enzymes (Xu et al., 2010a). Another experiment in prostate cancer has shown downregulation of mitochondrial antioxidant enzymes by miR-17-3p and subsequent enhancement of sensitivity of these cells to radiation (Xu et al., 2018).
Another study in multiple myeloma has shown that miR-17-5p regulates expression of the iron exporter ferroportin (FPN1), promote cell proliferation, enhance cell cycle progression, and suppress apoptosis. Expression of miR-17-5p is suppressed by the transcription factor Nrf2. Nrf2 also decreases FPN1 expression and enhanced accumulation of iron and production of ROS in the cells (Kong et al., 2019). miR-18a is another miRNA which is involved in the regulation of ferroptosis. This miRNA has been shown to suppress expression of ALOXE3 in glioblastoma cells. Besides, ALOXE3 knock-down has enhanced secretion of 12-HETE from glioblastoma cells, decreasing migration of these cells through activation of GsPCR/PI3K/Akt axis (Yang X. et al., 2021).
miR-20a has also been shown to regulate expression of FPN through binding to its 3′-UTR. Experiments in lung cancer cells have shown that down-regulation of FPN increases cell proliferation and colony formation, most probably through enhancing iron accessibility for neoplastic cells (Babu and Muckenthaler 2016).
In liver cancer cells, miR-18a has been shown to reduce expression of GCLC-a gene that regulates biosynthesis of glutathione. miR-18a also reduces GSH levels in tumor tissues (Anderton et al., 2017). Moreover, in this type of cancer, miR-22 targets TfR1 and inhibits cell cycle progression and growth (Greene et al., 2013). Besides, miR-152 is another miRNA that regulates ferroptosis in liver cancer cells (Huang et al., 2010). Another experiment in liver cancer cells shows the role of miR-503 in reduction of intracellular levels of SOD and glutathione .
The role of miRNAs in the regulation of ferroptosis has also been assessed in colorectal cancer cells. In this type of cancer, miR-24-2 levels has been inversely correlated with the levels of superoxide dismutase (SOD) (He et al., 2018). Moreover, induction of ROS by GT-094 has been found to be correlated with modulation of the miR-27a:ZBTB10-Sp1/Sp3/Sp4 axis (Pathi et al., 2011). miR-145 and miR-149 are two other miRNAs that affect expression of TFR1 and DMT1 in colorectal cancer cells (Hamara et al., 2013). Lung cancer is another type of cancer in which the role of miRNAs in the regulation of ferroptosis has been vastly investigated. For instance, miR-155 silencing has been shown to inhibit GST-π expression in A549/dox cells. miR-155 induces doxorubicin resistance via modulation of drug transportation and drug metabolism (Lv et al., 2016). miR-196a is another miRNA that has an indirect effect on ferrptosis. Suppression of this miRNA has suppressed stem cell self-renewal capacity, tumor growth and tumorigenicity through enhancement of expression of GPX3 (Liu et al., 2019). Moreover, miR-302a-3p has been found to induce ferroptosis in lung cancer cells via targeting ferroportin (Wei et al., 2021). Besides, miR-324-3p enhances cisplatin-induced ferroptosis in lung cancer cells .
Therefore, the effects of miRNAs on ferroptosis can be regarded as a mechanism for induction/prevention of different malignancies. Moreover, modulation of expression of ferroptosis-related miRNAs can be regarded as a potential treatment strategy for cancers. Table 1 shows the role of miRNAs in the regulation of ferroptosis in cancers.

miRNAs effects on ferroptosis in nonmalignant conditions
miRNAs have important roles in the ferroptosis in non-malignant conditions. Parkinson's disease is an example of disorders in which the role of miRNAs in the regulation of ferroptosis has been assessed. An experiment in this field has shown down-regulation of GPX4 in the animal model of this disorder in association with down-regulation of FTH1 and over-expression of miR-335. miR-335 mimic could decrease expression of FTH1, increase ferroptosis and facilitate progression of Parkinson's disease. Mechanistically, miR-335 targets 3′-UTR of FTH1. FTH1 silencing in 6-OHDA-induced cells has increased the proferroptosis impact of miR-335 and promoted pathologic events in the course of Parkinson's disease. In fact, miR-335 enhances ferroptosis via reduction of FTH1 and subsequent enhancement of iron release, lipid peroxidation and ROS buildup, while decreasing mitochondrial membrane potential . Figure 2 depicts this process.
Another experiment has shown aberrant expression of IL-6 and its receptor in cartilage samples of patients with intervertebral disc degeneration. Notably, IL-6 could down-regulate expression of miR-10a-5p, leading to derepression of IL-6R expression. IL-6 has a role in induction of ferroptosis in cartilage cells through stimulating oxidative stress and upsetting iron homeostasis. Up-regulation of miR-10a-5p could decrease IL-6R levels and attenuate IL-6associated ferroptosis to some extent (Bin et al., 2021).
Expression of miR-15a-5p has been shown to be increased in acute myocardial infarction. miR-15a-5p silencing has decreased mortality of myocardial cells in hypoxic conditions. Notably, GPX4 has been identified as the direct target of miR-15a-5p. Up-regulation of miR-15a-5p has enhanced ferroptosis and intensified myocardial cell damage during hypoxia. Knock-down of the transcription factor Egr-1 has led to down-regulation of miR-15a-5p, and subsequent up-regulation of GPX4, which results in reduction of ferroptosis and alleviation of myocardial damage (Fan et al., 2021).

FIGURE 1
Iron metabolism, antioxidant metabolism and lipid metabolism are the pivotal mechanisms in the ferroptosis process. The mentioned metabolismsrelated miRNAs network was represented by Cytoscape software. The common miRNAs (miR-675, miR-93, miR-27a, miR-34a and miR-141) in these three networks with the most degree and betweenness centrality as the key miRNAs are shown by green triangles (Luo et al., 2021a).

Frontiers in Molecular Biosciences
frontiersin.org miR-17-92 is another miRNA which modulates ferroptosis. This miRNA has been shown to protect endothelial cells from erastinassociated ferroptosis. In fact, over-expression of miR-17-92 can reduce erastin-associated growth suppression and ROS production in endothelial cells. Mechanistically, miR-17-92 exerts its effects through suppression of Zinc lipoprotein A20 expression. miR-17-92 up-regulation or A20 suppression has enhanced expression of ACSL4 endothelial cells (Xiao F. J. et al., 2019). Ferroptosis in animal model of intracerebral hemorrhage has been associated with reduced levels of miR-19b-3p and enhancement of IRP2 levels. Expression of IRP2 as a direct target of miR-19b-3p has been suppressed by miR-19b-3p mimictransfected adipose-derived stem cells. These exosomes could also attenuate hemin-associated cell damage and ferroptosis, thus improving neurologic function in the effected animals (Yi and Tang 2021). Taken together, ferroptosis-associated miRNAs are involved in the pathogenesis of a variety of non-malignant conditions, such as intervertebral disc degeneration, acute myocardial infarction, vascular diseases, intracerebral hemorrhage and preeclampsia. Table 2 shows the role of miRNAs in regulation of ferroptosis in non-malignant conditions.

Effects of different treatments on expression of ferroptosis-associated miRNAs
A number of drugs and treatments have been found to influence course of disorders through affecting expression of ferroptosis-associated miRNAs. For instance, experiments in animal models of intracerebral hemorrhage have shown that acupuncture can amend neuron cells death, inflammatory responses, and ferroptosis through downregulation of miR-23a-3p. The effects of acupuncture on alleviation of ferroptosis and reduction of miR-23a-3p expression have been verified by the observed enhancemnet of nuclear translocation of NFE2L2 and expression of heme oxygenase-1 and glutathione peroxidase 4 as well as reduction of iron and malondialdehyde levels and decrease in the accumulation of reactive oxygen species. Furthermore, antagomiR-23a-3p could inhibit the intracerebral hemorrhage-induced enhancemnet of Fluoro-Jade B-positive cells, production of proinflammatory cytokines, ferroptosis, and activity of NFE2L2. Mechanistically, miR-23a-3p has binding site on NFE2L2 (Kong et al., 2021). On the other hand, isorhynchophylline has been shown to ameliorate ferroptosis-associated nerve injury in the context of intracerebral hemorrhage through modulation of miR-122-5p (Zhao et al., 2021). Experiments in mouse hippocampal HT-22 cells exposed to ferric ammonium citrate alone or together with Isorhynchophylline have shown that Isorhynchophylline reduces the ferric ammonium citrate-associated cell injury. Isorhynchophylline also reduces the ferric ammonium citrateinduced reduction of miR-122-5p expression and ameliorates ferroptosis. Besides, miR-122-5p inhibitor could diminish the protective effect of Isorhynchophylline against ammonium citrate-associated ferroptosis in these cells. Mechanistically, miR-122-5p targets TP53, and restoration of TP53 attenuates the effect of miR-122-5p on ferroptotic markers and expression of SLC7A11. Taken together, miR-122-5p/TP53/SLC7A11 axis has been suggested as a potential mechanism in the etiology of intracranial hemorrhage (Zhao et al., 2021).
Moreover, metformin can induce ferroptosis of breast cancer cells through influencing expression of the GPX4 targeting miRNA miR-324-3p. Up-regulation of miR-324-3p has suppressed viability of breast cancer cells. In fact, metformin

FIGURE 2
The mechanism of ferroptosis induction in Parkinson's disease by miR-335. miR-335 targets FTH1 and degrades it to promote iron release, lipid peroxidation and reactive oxygen species (ROS) accumulation, and decreases mitochondrial membrane potential (MMP) and intensify ferroptosis and PD pathology. Although glutathione peroxidase 4 (GPX4) is not directly targeted by miR-335, up-regulation of miR-335 also leads to reduction of the levels of this ferroptosis marker protein .

Frontiers in Molecular Biosciences
frontiersin.org can be regarded as a potential anti-cancer agent via activation of ferroptosis . Similarly, lidocaine and levobupivacaine enhance ferroptosis of cancer cells through targeting miR-382-5p (Sun D. et al., 2021) and miR-489-3p (Mao et al., 2021), respectively. Table 3 shows the effects of different treatments on expression of ferroptosis-associated miRNAs.

Discussion
Several miRNAs have been found to affect ferroptosis through binding with 3′-UTR of genes participating in this process. GOT1, GPX2, GPX3, GPX4, FPN, GSH, GST, FTL, TFR1 and NRF2 are examples of ferroptosis-associated molecules which are regulated by miRNAs. Moreover, a number of miRNAs can affect ferroptosis through indirect routes. For instance, miR-152 can reduce expression of DNA methyltransferase one leading to global DNA hypomethylation and enhancement of expression of GSTP1 (Huang et al., 2010).
Since ferroptosis can eradicate cancer cells in an independent way from apoptosis , identification of the role of miRNAs in this process can propose new ways for combatting cancer progression. It is worth mentioning that some of abovementioned miRNAs that regulate ferroptosis, have additional roles in the regulation of other types of cell death. Thus, these miRNAs can induce cancer cell death from different routes.
Bioinformatics tools have facilitated identification of miRNAs with highest involvement in the ferroptosis, thus proposing the most appropriate targets for management of ferroptosis-associated disorders. A number of long non-coding RNAs and circRNAs have been found to affect expression of ferroptosis-related miRNAs. CircRHOT1, circABCB10, circRNA_000479, circFNDC3B, circIL4R, circ_0067934, circ-TTBK2, circ_0007142, circ_0013731, circKIF4A, circ0097009, lncOIP5-AS1, lncMT1DP and lncMEG8 have been recognized as competing endogenous RNAs for miRNAs that partake in the ferroptosis. Therefore, ferroptosis can be regulated by several members of non-coding RNAs.
Notably, acupuncture and a number of drugs such as physcion 8-O-β-glucopyranoside, isorhynchophylline, metformin, lidocaine and levobupivacaine have been shown to affect ferroptosis through modulation of miRNAs. Thus, identification of the role of miRNAs in the regulation of ferroptosis can facilitate design of novel therapeutic agents for treatment of diverse neoplastic or neurodegenerative disorders.
Based on the vast impact of ferroptosis on development of disorders, therapies targeting this process can be proposed as treatment modalities for several disorders including neoplastic and neurodegenerative disorders. Manipulation of expression of ferroptosis-associated miRNAs through different methods is regarded as a potential strategy to affect ferroptosis and intervene with the pathoetiology of mentioned disorders. Since ferroptosis might have opposite effects on the physiology of organs, context-based strategies are needed in this regard. Other issues that should be addressed before incorporation of miRNA-based therapies in the clinical settings are identification of safe and efficient methods for delivery of these kinds of therapies into the specific cells and monitoring the cellular response to these modalities.
Ferroptosis-related miRNAs can alter response of cancer cells to chemotherapeutic modalities. Therefore, manipulation of expression of these miRNAs not only affects the progression and evolution of cancer, but also influences the response to a variety of treatment options. In spite of extensive research on effectiveness of these modalities in cancer cell lines and animal models, there is no clinical trial for appraisal of these methods in the clinical settings. However, it is expected that combination of miRNA-based therapies with conventional or targeted anticancer therapies enhances the effectiveness of these therapies. Since cancer cells are heterogeneous in terms of miRNAs signature, it is necessary to have a miRNA profile for each patient before implementation of these novel methods in the clinical settings.

Author contributions
SG-F wrote the manuscript and revised it. SK, RM-L, and BH collected the data and designed the figures and tables. All authors read and approved the submitted version.

Funding
This study was financially supported by Grant from Medical School of Shahid Beheshti University of Medical Sciences.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.